A novel Affi-Cova magnetic nanoparticles for one-step covalent immobilization of His-tagged enzyme directly from crude cell lysate.

Owing to the rapid advancement of in vitro synthetic biology, functional carriers capable of covalently binding target proteins from crude lysates under mild conditions have garnered escalating attention. Herein, a magnetic nanoparticle with affinity/covalent bifunction (MNP@Affi-Cova) was developed for the direct covalent immobilization of the recombinant enzyme of His-tagged birA (r-birA) from crude cell lysates in a single step. This innovative approach is attributed to the presence of chelated Ni ions and epoxy groups on the surface of the beads.

Affinity-assisted covalent self-assembly of PduQ-SpyTag and Nox-SpyCatcher to construct multi-enzyme complexes on the surface of magnetic microsphere modified with chelated Ni.

It is of great significance to study the effect of multi-enzyme aggregation behavior at the interface on the formation of multi-enzyme complexes and their co-catalytic characteristics, which is helpful for us to design and construct immobilized multi-enzyme complex systems for in vitro synthetic biology. Here, a magnetic microsphere with chelated Ni, was prepared to explore the self-assembly characteristics of PduQ-SpyTag (P-T) and Nox-SpyCatcher (NC) on its surface, based on the mixed interaction mode consisting the affinity of His-tag/Ni and covalent binding of SpyTag/SpyCatcher. After studying the effect of saturated or unsaturated adsorption of P-T on the covalent binding between P-T and NC at the interface, a possible multienzyme interaction mechanism for the affinity-assisted covalent self-assembly on the Ni chelating surface was proposed.

Study on the properties of a dual-system-based protein scaffold for orthogonal self-assembly.

Protein scaffolds possessing the ability to efficiently organize enzymes to improve the catalytic performance, enzyme stability and provide an optimal micro-environment for biocatalysis. Here, SpyCatcher fused to the C-terminus of Treptavidin (a variant of streptavidin) to construct a chimeric tetramers protein scaffold (Tr-SC) with dual orthogonal conjugation moieties. The results showed that the expressed Tr-SC scaffold was an active tetramer with good stability under 80 °C and pH 6.

Performance of microenvironment-induced lipase immobilization on diversify surface of magnetic particle.

The orientation of the enzyme molecular on the interface of the carrier affects its activity. Therefore, it is very important to controllably induce the orientation of the enzyme on the surface to improve the performance of the immobilized enzyme. Magnetic nanoparticles were used to construct microenvironments with the different surface hydrophobicity and charge characteristics by controlled modification, and those particles with various microenvironments were further used to study their interaction with the lipase.